Peptidomimetic inhibitors of post-proline cleaving enzymes

ABSTRACT

Disclosed are inhibitors of post-proline cleavage enzymes, such as inhibitors of dipeptidyl peptidase IV, as well as pharmaceutical compositions thereof, and methods for using such inhibitors. In particular, the inhibitors are improved over those in the prior art by selection of particular classes of side chains in the P1 and/or P2 position of the inhibitor. The inhibitors can have a better therapeutic index, owing in part to reduced toxicity and/or improved specificity for the targeted protease.

BACKGROUND OF THE INVENTION

Proteases are enzymes that cleave proteins at single, specific peptidebonds. Proteases can be classified into four generic classes: serine,thiol or cysteinyl, acid or aspartyl, and metalloproteases (Cuypers etal., J. Biol. Chem. 257:7086 (1982)). Proteases are essential to avariety of biological activities, such as digestion, formation anddissolution of blood clots, reproduction and the immune reaction toforeign cells and organisms. Aberrant proteolysis is associated with anumber of disease states in man and other mammals. In many instances, itis beneficial to disrupt the function of one or more proteolytic enzymesin the course of therapeutically treating an animal.

The binding site for a peptide substrate consists of a series of“specificity subsites” across the surface of the enzyme. The term“specificity subsite” refers to a pocket or other site on the enzymecapable of interacting with a portion of a substrate for the enzyme. Indiscussing the interactions of peptides with proteases, e.g., serine andcysteine proteinases and the like, the present application utilizes thenomenclature of Schechter and Berger [(1967) Biochem. Biophys. Res.Commun. 27:157-162)]. The individual amino acid residues of a substrateor inhibitor are designated P1, P2, etc. and the corresponding subsitesof the enzyme are designated S1, S2, etc, starting with the carboxyterminal residue produced in the cleavage reaction. The scissile bond ofthe substrate is amide bond between S1-S1′ of the substrate. Thus, forthe peptide Xaa1-Xaa2-Xaa3-Xaa4 which is cleaved between the Xaa3 andXaa4 residues, the Xaa3 residue is referred to as the P1 residue andbinds to the S1 subsite of the enzyme, Xaa2 is referred to as the P2residue and binds to the S2 subsite, and so forth.

Dipeptidyl peptidase IV (DPIV), for example, is a serine protease whichcleaves N-terminal dipeptides from a peptide chain containing,preferably, a proline residue in the penultimate position, e.g., in theP1 position. DPIV belongs to a group of cell-membrane-associatedpeptidases and, like the majority of cell-surface peptidases, is a typeII integral membrane protein, being anchored to the plasma membrane byits signal sequence. DPIV is found in a variety of differentiatedmammalian epithelia, endothelia and hemapoetic cells and tissues,including those of lymphoid origin where it is found specifically on thesurface of CD4⁺ T cells. DPIV has been identified as the leukocytedifferentiation marker CD26.

SUMMARY OF THE INVENTION

One aspect of the invention provides a protease inhibitor represented byFormula I:

wherein

A represents a 3-8 membered heterocycle including the N and the Cαcarbon;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct;

R₁ represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group;

R₂ is absent or represents one or more substitutions to the ring A, eachof which can independently be a halogen, a lower alkyl, a lower alkenyl,a lower alkynyl, a carbonyl, a thiocarbonyl, an amino, an acylamino, anamido, a cyano, a nitro, an azido, a sulfate, a sulfonate, asulfonamido, —(CH₂)_(m)—R₆, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₆, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)R₆;

R_(3a) represents a hydrogen or a substituent which does not conjugatethe electron pair of the nitrogen from which it pends;

R_(3b) is absent, or represents a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R_(4a) and R_(4b) each independently represent a hydrogen, lower alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl,carboxyl, carboxamide, carbonyl, or cyano, with the caveat that eitherboth or neither of R_(4a) and R_(4b) are hydrogen;

R_(4c) represents a halogen, an amine, an alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl, carboxyl,carboxamide, carbonyl, or cyano;

R₆ represents, independently for each occurrence, an aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety;

z is zero or an integer in the range of 1 to 3; m is zero or an integerin the range of 1 to 8; and n is an integer in the range of 1 to 8.

Another aspect of the invention provides a protease inhibitorrepresented by Formula III:

wherein

R represents hydrogen, a halogen, or a branched or unbranched C1-C6alkyl;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct;

R₁ represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group;

R_(3a) represents a hydrogen or a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R_(3b) is absent, or represents a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R_(4a) and R_(4b) each independently represent a hydrogen, lower alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl,carboxyl, carboxamide, carbonyl, or cyano, with the caveat that eitherboth or neither of R_(4a) and R_(4b) are hydrogen;

R_(4c) represents a halogen, an amine, an alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl, carboxyl,carboxamide, carbonyl, or cyano; and

z is zero or an integer in the range of 1 to 3.

Yet another aspect of the invention provides a protease inhibitorrepresented by Formula IV:

wherein

A represents a 3-8 membered heterocycle including the N and the Cαcarbon;

B represents a C3-C8 ring, or C7-C14 fused bicyclic or tricyclic ringsystem;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct;

R₁ represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group;

R₂ is absent or represents one or more substitutions to the ring A, eachof which can independently be a halogen, a lower alkyl, a lower alkenyl,a lower alkynyl, a carbonyl, a thiocarbonyl, an amino, an acylamino, anamido, a cyano, a nitro, an azido, a sulfate, a sulfonate, asulfonamido, —(CH₂)_(m)—R₆, —(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl,—(CH₂)_(m)—O-lower alkenyl, —(CH₂)_(n)—O—(CH₂)_(m)—R₆, —(CH₂)_(m)—SH,—(CH₂)_(m)—S-lower alkyl, —(CH₂)_(m)—S-lower alkenyl,—(CH₂)_(n)—S—(CH₂)_(m)—R₆;

R_(3b) is absent, or represents a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R₆ represents, independently for each occurrence, an aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

Still another aspect of the invention relates to a protease inhibitorrepresented by Formula VI:

wherein

B represents a C3-C8 ring, or C7-C14 fused bicyclic or tricyclic ringsystem;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct;

R represents hydrogen, a halogen, or a branched or unbranched C1-C6alkyl;

R₁ represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group; and

R_(3b) is absent, or represents a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl.

In certain preferred embodiments, the W represents —CN, —CH═NR₅,

wherein,

Y₁ and Y₂ each independently represent —OH, or a group capable of beinghydrolyzed to a hydroxyl group, including cyclic derivatives where Y₁and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure;

R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,—(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH, —(CH₂)n-S-alkyl,—(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl, —(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂,—C(O)C(O)OR₇;

R₆ represents, independently for each occurrence, an aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety;

R₇ represents, independently for each occurrence, hydrogen, or an alkyl,alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle moiety;

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In certain preferred embodiments of the inhibitors, W represents:

wherein, Y₁, Y₂, R₅ are as defined above.

In certain preferred embodiments, W represents —B(OH)₂, or a prodrugthereof which is hydrolyzed to —B(OH)₂ in vivo.

In certain other preferred embodiments, W represents —C(═O)—R₅, whereinR₅ is a hydrogen or —C(X₁)(X₂)X₃, wherein X₁ is a fluorine, and X₂ andX₃, if halogens, are also fluorine.

In certain embodiments of the inhibitors, R_(4a), R_(4b) and R_(4c) eachindependently represent a small hydrophobic group, such as selected fromthe group consisting of halogens, lower alkyls, lower alkenyls, andlower alkynyls.

In certain embodiments of the inhibitors, R_(4a) and R_(4b) eachrepresent hydrogen, and R_(4c) represents a small hydrophobic group.

In certain embodiments of the inhibitors, R_(4a) and R_(4b) eachrepresent hydrogen, and R_(4c) represents a cycloalkyl,heterocycloalkyl, aryl, or heteroaryl, and in certain preferredembodiments, is a C3-C8 cycloalkyl.

In certain embodiments of the inhibitors, R₂ is absent, or represents—OH.

In certain embodiments of the inhibitors, R_(3a) a hydrogen and R_(3b)is absent.

In certain embodiments of the inhibitors, R₁ is an amino acid residue ora peptidyl moiety which is a substrate for a protease.

In certain embodiments of the inhibitors, the protease inhibitorinhibits DPIV with a Ki of 50 nm or less.

In certain embodiments of the inhibitors, the inhibitor is orallyactive.

In certain embodiments of the inhibitors, the inhibitor has atherapeudic index in humans of at least 2, and even more preferably 5,10 or even 100, e.g., such as a therapeudic index for regulating glucosemetabolism.

Another aspect of the invention provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and one or more of thesubject protease inhibitors, or a pharmaceutically acceptable salt orprodrug thereof.

Another aspect of the invention provides for use of one or more of thesubject inhibitors in the manufacture of a medicament for inhibiting apost-proline cleaving enzyme in vivo. For example, the subjectinhibitors can be used to manufacture medicaments for increasing plasmaconcentrations of one or peptide hormones processed by post-prolinecleaving enzymes (e.g., DP-IV and the like). Exemplary medicaments areuseful in increasing plasma concentrations of such hormones asglucagons-like peptide, NPY, PPY, secretin, GLP-1, GLP-2, and GIP.

In certain preferred embodiments, the subject inhibitors can be used tomanufacture medicaments for regulating glucose metabolism, such as foruse in treating patients suffering from Type II diabetes, insulinresistance, glucose intolerance, hyperglycemia, hypoglycemia,hyperinsulinemia, obesity, hyperlipidemia, or hyperlipoproteinemia.

Yet another aspect of the invention provides a packaged pharmaceuticalcomprising: a preparation of one or more of the subject proteaseinhibitor; a pharmaceutically acceptable carrier; and instructions,written and/or pictorial, describing the use of the preparation forinhibiting a post-proline cleaving enzyme in vivo, such as forregulating glucose metabolism.

The packaged pharmaceutical can also include, e.g., as co-formulationthe protease inhibitor or simply co-packaged, insulin and/or aninsulinotropic agent.

The packaged pharmaceutical can also include, e.g., as co-formulationthe protease inhibitor or simply co-packaged, an M1 receptor antagonist,a prolactin inhibitor, agents acting on the ATP-dependent potassiumchannel of β-cells, metformin, and/or glucosidase inhibitors.

The present invention also relates to improved methods for the long-termreduction and abatement of at least one of the foregoing disorders basedon a therapeutic regimen administered over the short-term.

The present invention further provides a method for regulating, andaltering on a long-term basis, the glucose and lipogenic responses ofvertebrate animals, including humans.

In particular, the compounds of the invention may be employed to providemethods for producing long lasting beneficial changes in one or more ofthe following: the sensitivity of the cellular response of a species toinsulin (reduction of insulin resistance), blood insulin levels,hyperinsulinemia, blood glucose levels, the amount of body fat stores,blood lipoprotein levels, and thus to provide effective treatments fordiabetes, obesity and/or atherosclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic representation of the synthesis of aCyclohexylglycine-boro-Ala.

FIG. 2 is a blood glucose values curve during oral glucose challengetest in zucker rats following oral administration ofCyclohexylglycine-boro-Ala.

FIG. 3 is a time course of inactivation curve of His-boro-Ala at pH 8.

FIG. 4 is a time course of inactivation curve of Ala-boro-Ala at pH 8.

FIG. 5 is a time course of inactivation curve of Phg-boro-Ala at pH 8.

FIG. 6 is a time course of inactivation curve ofCyclohexylglycine-boro-Ala at pH 8.

FIG. 7 is a bar graph illustrating DPPIV enzyme activity as measuredfrom rat serum samples before and 1 hour after administration ofCyclohexylglycine-boro-Ala.

FIG. 8 is a diagrammatic representation of the conformation equilibriumof Xaa-boro-Alanine compounds.

FIG. 9 is the UV chromatograph of t-Butyl-glycine-Pro-nitrile.

FIG. 10 is a graph showing the DPIV inhibitory activity ofcyclohexylalanine-boroPro,

FIG. 11 is a graph showing the DPIV inhibitory activity of1,2,3,4-tetrahydroisoquinoline-boroProline,

FIG. 12 is a graph showing the DPIV inhibitory activity of1,2,3,4-tetrahydro-beta-carboline-boroProline,

FIG. 13 are two graphs, MS and NMR, showing the purification ofEthylglycine-2-boroThiazolidine,

FIG. 14 are two graphs, UV and MS, showing the purification ofEthylglycine-boroHydroxyproline,

FIG. 15 are two graphs, UV and MS, showing the purification ofdiaminoglycine-boroProline,

FIG. 16 is a graph showing the DPIV inhibitory activity ofEthylglycine-N(methyl)boroAlanine,

FIG. 17 is a graph showing the DPIV inhibitory activity ofEthylglycine-boroPiperidine,

FIG. 18 is an NMR spectra for Ethylglycine-N(methyl)boroGlycine,

FIG. 19 is an NMR spectra for t-butylglycine-boroAlanine,

FIG. 20 is a graph showing the in vivo DPIV inhibitory activity, at 0.05mg/kg, of t-butylglycine-boroProline,

FIG. 21 is a graph showing the in vivo DPIV inhibitory activity, at 0.05mg/kg, of isopropylglycine-boroProline,

FIG. 22 is a graph showing the in vivo DPIV inhibitory activity, at 0.05mg/kg, of ethylglycine-boroProline,

FIG. 23 is a graph showing the in vivo DPIV inhibitory activity, at 0.05mg/kg, of (allo)isoleucine-boroProline,

DETAILED DESCRIPTION I. Overview

The present invention relates to inhibitors of post-proline cleavingenzymes, such as inhibitors of dipeptidyl peptidase IV, as well aspharmaceutical compositions thereof, and methods for using suchinhibitors. In particular, the inhibitors of the present invention areimproved over those in the prior art by selection of particular classesof sidechains in the P1 and/or P2 position of the inhibitor. Salientfeatures for compounds of the present invention include: bettertherapeutic indices, owing in part to reduced toxicity and/or improvedspecificity for the targeted protease; better oral availability;increased shelf-life; and/or increased duration of action (such assingle oral dosage formulations which are effective for more than 4hours, and even more preferably for more 8, 12 or 16 hours).

The compounds of the present invention can be used as part of treatmentsfor a variety of disorders/conditions, such as those which are mediatedby DPIV. For instance, the subject inhibitors can be used to up-regulateGIP and GLP-1 activities, e.g., by increasing the half-life of thosehormones, as part of a treatment for regulating glucose levels and/ormetabolism, e.g., to reduce insulin resistance, treat hyperglycemia,hyperinsulinemia, obesity, hyperlipidemia, hyperlipoprotein-emia (suchas chylomicrons, VLDL and LDL), and to regulate body fat and moregenerally lipid stores, and, more generally, for the improvement ofmetabolism disorders, especially those associated with diabetes, obesityand/or atherosclerosis.

While not wishing to bound by any particular theory, it is observed thatcompounds which inhibit DPIV are, correlatively, able to improve glucosetolerance (See Examples 2 and 4), though not necessarily throughmechanisms involving DPIV inhibition per se. Indeed, the applicant haspreviously demonstrated an effect in mice lacking a GLP-1 receptorsuggesting that the subject method may not include a mechanism of actiondirectly implicating GLP-1 itself, though it has not been ruled out thatGLP-1 may have other receptors. However, in light of the correlationwith DPIV inhibition, in preferred embodiments, the subject methodutilizes an agent with a Ki for DPIV inhibition of 50.0 nm or less, morepreferably of 10.0 nm or less, and even more preferably of 1.0, 0.1 oreven 0.01 nM or less. Indeed, inhibitors with Ki values in the picomolarand even femtomolar range are contemplated. Thus, while the activeagents are described herein, for convenience, as “DPIV inhibitors”, itwill be understood that such nomenclature is not intending to limit thesubject invention to a particular mechanism of action.

Certain of the subject compounds have extended duration. Accordingly, incertain preferred embodiments, the inhibitor(s) is selected, and theamount of inhibitor formulated, to provide a dosage which inhibits serumPPCE (e.g., DPIV) levels by at least 50 percent for at least 4 hoursafter a single dose, and even more preferably for at least 8 hours oreven 12 or 16 hours after a single dose.

For instance, in certain embodiments the method involves administrationof a DPIV inhibitor, preferably at a predetermined time(s) during a24-hour period, in an amount effective to improve one or more aberrantindices associated with glucose metabolism disorders (e.g., glucoseintolerance, insulin resistance, hyperglycemia, hyperinsulinemia andType I and II diabetes).

In other embodiments, the method involves administration of a DPIVinhibitor in an amount effective to improve aberrant indices associatedwith obesity. Fat cells release the hormone leptin, which travels in thebloodstream to the brain and, through leptin receptors there, stimulatesproduction of GLP-1. GLP-1, in turn, produces the sensation of beingfull. The leading theory is that the fat cells of most obese peopleprobably produce enough leptin, but leptin may not be able to properlyengage the leptin receptors in the brain, and so does not stimulateproduction of GLP-1. There is accordingly a great deal of researchtowards utilizing preparations of GLP-1 as an appetite suppressant. Thesubject method provides a means for increasing the half-life of bothendogenous and ectopically added GLP-1 in the treatment of disordersassociated with obesity.

In a more general sense, the present invention provides methods andcompositions for altering the pharmokinetics of a variety of differentpolypeptide hormones by inhibiting the proteolysis of one or morepeptide hormones by DPIV or some other proteolytic activity.Post-secretory metabolism is an important element in the overallhomeostasis of regulatory peptides, and the other enzymes involved inthese processes may be suitable targets for pharmacological interventionby the subject method.

For example, the subject method can be used to increase the half-life ofother proglucagon-derived peptides, such as glicentin (corresponding toPG 1-69), oxyntomodulin (PG 33-69), glicentin-related pancreaticpolypeptide (GRPP, PG 1-30), intervening peptide-2 (IP-2, PG111-122amide), and glucagon-like peptide-2 (GLP-2, PG 126-158).

GLP-2, for example, has been identified as a factor responsible forinducing proliferation of intestinal epithelium. See, for example,Drucker et al. (1996) PNAS 93:7911. The subject method can be used aspart of a regimen for treating injury, inflammation or resection ofintestinal tissue, e.g., where enhanced growth and repair of theintestinal mucosal epithelial is desired, such as in the treatment ofChron's disease or Inflammatory Bowel Disease (IBD).

DPIV has also been implicated in the metabolism and inactivation ofgrowth hormone-releasing factor (GHRF). GHRF is a member of the familyof homologous peptides that includes glucagon, secretin, vasoactiveintestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitaryadenylate cyclase activating peptide (PACAP), gastric inhibitory peptide(GIP) and helodermin. Kubiak et al. (1994) Peptide Res 7:153. GHRF issecreted by the hypothalamus, and stimulates the release of growthhormone (GH) from the anterior pituitary. Thus, the subject method canbe used to improve clinical therapy for certain growth hormone deficientchildren, and in clinical therapy of adults to improve nutrition and toalter body composition (muscle vs. fat). The subject method can also beused in veterinary practice, for example, to develop higher yield milkproduction and higher yield, leaner livestock.

Likewise, the DPIV inhibitors of the subject invention can be used toalter the plasma half-life of secretin, VIP, PHI, PACAP, GIP and/orhelodermin. Additionally, the subject method can be used to alter thepharmacokinetics of Peptide YY and neuropeptide Y, both members of thepancreatic polypeptide family, as DPIV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity.

In other embodiments, the subject inhibitors can be used to stimulatehematopoiesis.

In still other embodiments, the subject inhibitors can be used toinhibit growth or vascularization of transformed cells/tissues, e.g., toinhibit cell proliferation such as that associated with tumor growth andmetastasis, and for inhibiting angiogenesis in an abnormal proliferativecell mass.

In yet other embodiments, the subject inhibitors can be used to reduceimmunological responses, e.g., as an immunosuppressant.

In yet other examples, the DPIV inhibitors according to the presentinvention can be used to treat CNS maladies such as strokes, tumors,ischemia, Parkinson's disease, memory loss, hearing loss, vision loss,migraines, brain injury, spinal cord injury, Alzheimer's disease andamyotrophic lateral sclerosis (which has a CNS component). Additionally,the DPIV inhibitors can be used to treat disorders having a moreperipheral nature, including multiplesclerosis and diabetic neuropathy.

Another aspect of the present invention relates to pharmaceuticalcompositions of the subject post-proline cleaving enzyme inhibitors,particularly DPIV inhibitors, and their uses in treating and/orpreventing disorders which can be improved by altering the homeostasisof peptide hormones. In a preferred embodiment, the inhibitors havehypoglycemic and antidiabetic activities, and can be used in thetreatment of disorders marked by aberrant glucose metabolism (includingstorage). In particular embodiments, the compositions of the subjectmethods are useful as insulinotropic agents, or to potentiate theinsulinotropic effects of such molecules as GLP-1. In this regard,certain embodiments of the present compositions can be useful for thetreatment and/or prophylaxis of a variety of disorders, including one ormore of: hyperlipidemia, hyperglycemia, obesity, glucose toleranceinsufficiency, insulin resistance and diabetic complications.

In general, the inhibitors of the subject method will be smallmolecules, e.g., with molecular weights less than 7500 amu, preferablyless than 5000 amu, and even more preferably less than 2000 or even 1000amu. In preferred embodiments, the inhibitors will be orally active.

II. Definitions

The term “high affinity” as used herein means strong binding affinitybetween molecules with a dissociation constant K_(D) of no greater than1 μM. In a preferred case, the K_(D) is less than 100 nM, 10 nM, 1 nM,100 pM, or even 10 pM or less. In a most preferred embodiment, the twomolecules can be covalently linked (K_(D) is essentially 0).

The term “boro-Ala” refers to the analog of alanine in which thecarboxylate group (COOH) is replaced with a boronyl group (B(OH)₂).Likewise, the term “boro-Pro” refers to the analog of praline in whichthe carboxylate group (COOH) is replaced with a boronyl group (B(OH)₂).More generally, the term “boro-Xaa”, where Xaa is an amino acid residue,refers to the analog of an amino acid in which the carboxylate group(COOH) is replaced with a boronyl group (B(OH)₂).

A “patient” or “subject” to be treated by the subject method can meaneither a human or non-human subject.

The term “ED₅₀” means the dose of a drug that, in 50% of patients, willprovide a clinically relevant improvement or change in a physiologicalmeasurement, such as glucose responsiveness, increase in hematocrit,decrease in tumor volume, etc.

The term “IC₅₀” means the dose of a drug that inhibits a biologicalactivity by 50%, e.g., the amount of inhibitor required to inhibit atleast 50% of DPIV (or other PPCE) activity in vivo.

A compound is said to have an “insulinotropic activity” if it is able tostimulate, or cause the stimulation of, the synthesis or expression ofthe hormone insulin.

The term “interact” as used herein is meant to include all interactions(e.g., biochemical, chemical, or biophysical interactions) betweenmolecules, such as protein-protein, protein-nucleic acid, nucleicacid-nucleic acid, protein-small molecule, nucleic acid-small moleculeor small molecule-small molecule interactions.

The term “LD₅₀” means the dose of a drug that is lethal in 50% of testsubjects.

The term “prophylactic or therapeutic” treatment is art-recognized andincludes administration to the host of one or more of the subjectcompositions. If it is administered prior to clinical manifestation ofthe unwanted condition (e.g., disease or other unwanted state of thehost animal) then the treatment is prophylactic, i.e., it protects thehost against developing the unwanted condition, whereas if it isadministered after manifestation of the unwanted condition, thetreatment is therapeutic, (i.e., it is intended to diminish, ameliorate,or stabilize the existing unwanted condition or side effects thereof).

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “therapeutic index” refers to the therapeutic index of a drugdefined as LD₅₀/ED₅₀.

A “therapeutically effective amount” of a compound, e.g., such as a DPIVinhibitor of the present invention, with respect to the subject methodof treatment, refers to an amount of the compound(s) in a preparationwhich, when administered as part of a desired dosage regimen (to amammal, preferably a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment.

A “single oral dosage formulation” is a dosage which provides an amountof drug to produce a serum concentration at least as great as the EC₅₀for that drug, but less than the LD₅₀. Another measure for a single oraldosage formulation is that it provides an amount of drug necessary toproduce a serum concentration at least as great as the IC₅₀ for thatdrug, but less than the LD₅₀. By either measure, a single oral dosageformulation is preferably an amount of drug which produces a serumconcentration at least 10 percent less than the LD₅₀, and even morepreferably at least 50 percent, 75 percent or even 90 percent less thanthe drug's the LD₅₀.

An aliphatic chain comprises the classes of alkyl, alkenyl and alkynyldefined below. A straight aliphatic chain is limited to unbranchedcarbon chain radicals. As used herein, the term “aliphatic group” refersto a straight chain, branched-chain, or cyclic aliphatic hydrocarbongroup and includes saturated and unsaturated aliphatic groups, such asan alkyl group, an alkenyl group, and an alkynyl group.

Alkyl refers to a fully saturated branched or unbranched carbon chainradical having the number of carbon atoms specified, or up to 30 carbonatoms if no specification is made. For example, alkyl of 1 to 8 carbonatoms refers to radicals such as methyl, ethyl, propyl, butyl, pentyl,hexyl, heptyl, and octyl, and those radicals which are positionalisomers of these radicals. Alkyl of 10 to 30 carbon atoms includesdecyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyland tetracosyl. In preferred embodiments, a straight chain or branchedchain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C₁-C₃₀for straight chains, C₃-C₃₀ for branched chains), and more preferably 20or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms intheir ring structure, and more preferably have 5, 6 or 7 carbons in thering structure.

Moreover, the term “alkyl” (or “lower alkyl”) as used throughout thespecification, examples, and claims is intended to include both“unsubstituted alkyls” and “substituted alkyls”, the latter of whichrefers to alkyl moieties having substituents replacing a hydrogen on oneor more carbons of the hydrocarbon backbone. Such substituents caninclude, for example, a halogen, a hydroxyl, a carbonyl (such as acarboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (suchas a thioester, a thioacetate, or a thioformate), an alkoxyl, aphosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, anamido, an amidine, a cyano, a nitro, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude substituted and unsubstituted forms of amino, azido, imino,amido, phosphoryl (including phosphonate and phosphinate), sulfonyl(including sulfate, sulfonamido, sulfamoyl and sulfonate), and silylgroups, as well as ethers, alkylthios, carbonyls (including ketones,aldehydes, carboxylates, and esters), —CF₃, —CN and the like. Exemplarysubstituted alkyls are described below. Cycloalkyls can be furthersubstituted with alkyls, alkenyls, alkoxyls, alkylthios, aminoalkyls,carbonyl-substituted alkyls, —CF₃, —CN, and the like.

Unless the number of carbons is otherwise specified, “lower alkyl”, asused herein, means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl, and tert-butyl. Likewise, “lower alkenyl” and“lower alkynyl” have similar chain lengths. Throughout the application,preferred alkyl groups are lower alkyls. In preferred embodiments, asubstituent designated herein as alkyl is a lower alkyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —(S)-alkyl, —(S)-alkenyl,—(S)-alkynyl, and —(S)—(CH₂)_(m)—R₁, wherein m and R₁ are defined below.Representative alkylthio groups include methylthio, ethylthio, and thelike.

Alkenyl refers to any branched or unbranched unsaturated carbon chainradical having the number of carbon atoms specified, or up to 26 carbonatoms if no limitation on the number of carbon atoms is specified; andhaving 1 or more double bonds in the radical. Alkenyl of 6 to 26 carbonatoms is exemplified by hexenyl, heptenyl, octenyl, nonenyl, decenyl,undecenyl, dodenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl,heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, heneicosoenyl,docosenyl, tricosenyl and tetracosenyl, in their various isomeric forms,where the unsaturated bond(s) can be located anywhere in the radical andcan have either the (Z) or the (E) configuration about the doublebond(s).

Alkynyl refers to hydrocarbyl radicals of the scope of alkenyl, buthaving 1 or more triple bonds in the radical.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined below, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynyl, —O—(CH₂)_(m)—R₁,where m and R₁ are described below.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formulae:

wherein R₃, R₅ and R₆ each independently represent a hydrogen, an alkyl,an alkenyl, —(CH₂)_(m)—R₁, or R₃ and R₅ taken together with the N atomto which they are attached complete a heterocycle having from 4 to 8atoms in the ring structure; R₁ represents an alkenyl, aryl, cycloalkyl,a cycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or aninteger in the range of 1 to 8. In preferred embodiments, only one of R₃or R₅ can be a carbonyl, e.g., R₃, R₅ and the nitrogen together do notform an imide. In even more preferred embodiments, R₃ and R₅ (andoptionally R₆) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R₁. Thus, the term “alkylamine” as used hereinmeans an amine group, as defined above, having a substituted orunsubstituted alkyl attached thereto, i.e., at least one of R₃ and R₅ isan alkyl group. In certain embodiments, an amino group or an alkylamineis basic, meaning it has a pK_(a)≧7.00. The protonated forms of thesefunctional groups have pK_(a)s relative to water above 7.00.

The term “carbonyl” is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₇represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₁ or apharmaceutically acceptable salt, R₈ represents a hydrogen, an alkyl, analkenyl or —(CH₂)_(m)—R₁, where m and R₁ are as defined above. Where Xis an oxygen and R₇ or R₈ is not hydrogen, the formula represents an“ester”. Where X is an oxygen, and R₇ is as defined above, the moiety isreferred to herein as a carboxyl group, and particularly when R₇ is ahydrogen, the formula represents a “carboxylic acid”. Where X is anoxygen, and R₈ is hydrogen, the formula represents a “formate”. Ingeneral, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R₇ or R₈ is not hydrogen, the formula represents a“thioester” group. Where X is a sulfur and R₇ is hydrogen, the formularepresents a “thiocarboxylic acid” group. Where X is a sulfur and R₈ ishydrogen, the formula represents a “thioformate” group. On the otherhand, where X is a bond, and R₇ is not hydrogen, the above formularepresents a “ketone” group. Where X is a bond, and R₇ is hydrogen, theabove formula represents an “aldehyde” group.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl,sulfamoyl, sulfinyl, ether, alkylthio, sulfonyl, ketone, aldehyde,ester, a heterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN,or the like.

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described herein above. The permissible substituentscan be one or more and the same or different for appropriate organiccompounds. For purposes of this invention, the heteroatoms such asnitrogen may have hydrogen substituents and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds.

The term “hydrocarbyl” refers to a monovalent hydrocarbon radicalcomprised of carbon chains or rings of up to 26 carbon atoms to whichhydrogen atoms are attached. The term includes alkyl, cycloalkyl,alkenyl, alkynyl and aryl groups, groups which have a mixture ofsaturated and unsaturated bonds, carbocyclic rings and includescombinations of such groups. It may refer to straight chain,branched-chain, cyclic structures or combinations thereof.

The term “hydrocarbylene” refers to a divalent hydrocarbyl radical.Representative examples include alkylene, phenylene, or cyclohexylene.Preferably, the hydrocarbylene chain is fully saturated and/or has achain of 1-10 carbon atoms.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

The term “sulfamoyl” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₃ and R₅ are as defined above.

The term “sulfate” is art recognized and includes a moiety that can berepresented by the general formula:

in which R₇ is as defined above.

The term “sulfonamido” is art recognized and includes a moiety that canbe represented by the general formula:

in which R₂ and R₄ are as defined above.

The term “sulfonate” is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₇ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula:

in which R₁₂ is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups toproduce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

A “small” substituent is one of 10 atoms or less.

By the terms “amino acid residue” and “peptide residue” is meant anamino acid or peptide molecule without the —OH of its carboxyl group. Ingeneral the abbreviations used herein for designating the amino acidsand the protective groups are based on recommendations of the IUPAC-IUBCommission on Biochemical Nomenclature (see Biochemistry (1972)11:1726-1732). For instance Met, Ile, Leu, Ala and Gly represent“residues” of methionine, isoleucine, leucine, alanine and glycine,respectively. By the residue is meant a radical derived from thecorresponding α-amino acid by eliminating the OH portion of the carboxylgroup and the H portion of the α-amino group. The term “amino acid sidechain” is that part of an amino acid exclusive of the —CH(NH₂)COOHportion, as defined by K. D. Kopple, “Peptides and Amino Acids”, W. A.Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33; examples ofsuch side chains of the common amino acids are —CH₂CH₂SCH₃ (the sidechain of methionine), —CH₂(CH₃)—CH₂CH₃ (the side chain of isoleucine),—CH₂CH(CH₃)₂ (the side chain of leucine) or H— (the side chain ofglycine).

For the most part, the amino acids used in the application of thisinvention are those naturally occurring amino acids found in proteins,or the naturally occurring anabolic or catabolic products of such aminoacids which contain amino and carboxyl groups. Particularly suitableamino acid side chains include side chains selected from those of thefollowing amino acids: glycine, alanine, valine, cysteine, leucine,isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid,glutamine, asparagine, lysine, arginine, proline, histidine,phenylalanine, tyrosine, and tryptophan, and those amino acids and aminoacid analogs which have been identified as constituents ofpeptidylglycan bacterial cell walls.

The term amino acid residue further includes analogs, derivatives andcongeners of any specific amino acid referred to herein, as well asC-terminal or N-terminal protected amino acid derivatives (e.g. modifiedwith an N-terminal or C-terminal protecting group). For example, thepresent invention contemplates the use of amino acid analogs wherein aside chain is lengthened or shortened while still providing a carboxyl,amino or other reactive precursor functional group for cyclization, aswell as amino acid analogs having variant side chains with appropriatefunctional groups). For instance, the subject compound can include anamino acid analog such as, for example, cyanoalanine, canavanine,djenkolic acid, norleucine, 3-phosphoserine, homoserine,dihydroxy-phenylalanine, 5-hydroxytryptophan, 1-methylhistidine,3-methylhistidine, diaminopimelic acid, ornithine, or diaminobutyricacid. Other naturally occurring amino acid metabolites or precursorshaving side chains which are suitable herein will be recognized by thoseskilled in the art and are included in the scope of the presentinvention.

Also included are the (D) and (L) stereoisomers of such amino acids whenthe structure of the amino acid admits of stereoisomeric forms. Theconfiguration of the amino acids and amino acid residues herein aredesignated by the appropriate symbols (D), (L) or (DL), furthermore whenthe configuration is not designated the amino acid or residue can havethe configuration (D), (L) or (DL). It will be noted that the structureof some of the compounds of this invention includes asymmetric carbonatoms. It is to be understood accordingly that the isomers arising fromsuch asymmetry are included within the scope of this invention. Suchisomers can be obtained in substantially pure form by classicalseparation techniques and by sterically controlled synthesis. For thepurposes of this application, unless expressly noted to the contrary, anamed amino acid shall be construed to include both the (D) or (L)stereoisomers.

The phrase “protecting group” as used herein means substituents whichprotect the reactive functional group from undesirable chemicalreactions. Examples of such protecting groups include esters ofcarboxylic acids and boronic acids, ethers of alcohols and acetals andketals of aldehydes and ketones. For instance, the phrase “N-terminalprotecting group” or “amino-protecting group” as used herein refers tovarious amino-protecting groups which can be employed to protect theN-terminus of an amino acid or peptide against undesirable reactionsduring synthetic procedures. Examples of suitable groups include acylprotecting groups such as, to illustrate, formyl, dansyl, acetyl,benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl; aromaticurethane protecting groups as, for example, benzyloxycarbonyl (Cbz); andaliphatic urethane protecting groups such as t-butoxycarbonyl (Boc) or9-Fluorenylmethoxycarbonyl (FMOC).

As noted above, certain compounds of the present invention may exist inparticular geometric or stereoisomeric forms. The present inventioncontemplates all such compounds, including cis- and trans-isomers, R-and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term “hydrocarbon” is contemplatedto include all permissible compounds having at least one hydrogen andone carbon atom. In a broad aspect, the permissible hydrocarbons includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

A compound is said to have an “insulinotropic activity” if it is able tostimulate, or cause the stimulation of, the synthesis or expression ofthe hormone insulin.

It will be understood that all generic structures recited herein, withrespect to appropriate combinations of substituents, are intended tocover those embodiments permitted by valency and stability.

III. Exemplary Embodiments

(i). Compounds

One aspect of the present invention is a compound represented by FormulaI:

wherein

A represents a 3-8 membered heterocycle including the N and the Cαcarbon;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct, as for example, —CN,—CH═NR₅,

R₁ represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group;

R₂ is absent or represents one or more substitutions to the ring A, eachof which can independently be a halogen, a lower alkyl, a lower alkenyl,a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, ora ketone), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an amino, an acylamino, an amido, a cyano, a nitro, anazido, a sulfate, a sulfonate, a sulfonamido, —(CH₂)_(m)—R₆,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)R₆, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₆;

R_(3a) represents a hydrogen or a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R_(3b) is absent, or represents a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R_(4a) and R_(4b) each independently represent a hydrogen, lower alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl,carboxyl, carboxamide, carbonyl, or cyano, with the caveat that eitherboth or neither of R_(4a) and R_(4b) are hydrogen;

R_(4c) represents a halogen, an amine, an alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl, carboxyl,carboxamide, carbonyl, or cyano;

R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,—(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH, —(CH₂)n-S-alkyl,—(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl, —(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂,—C(O)C(O)OR₇;

R₆ represents, independently for each occurrence, an aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety;

R₇ represents, independently for each occurrence, hydrogen, or an alkyl,alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle moiety;and

Y₁ and Y₂ each independently represent —OH, or a group capable of beinghydrolyzed to a hydroxyl group, including cyclic derivatives where Y₁and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen;

z is zero or an integer in the range of 1 to 3 (preferably 0 or 1); m iszero or an integer in the range of 1 to 8; and n is an integer in therange of 1 to 8.

In certain embodiments, the protease inhibitor is represented in thegeneral formula II:

where R₁, R_(3a), R_(3b), R_(4a), R_(4b), R_(4c) and W are as definedabove, and p is an integer from 1 to 3. In certain preferredembodiments, p is 1, and R_(3a) is a hydrogen and R_(3b) is absent.

Another aspect of the present invention is a compound represented byFormula III:

wherein

R represents hydrogen, a halogen, or a branched or unbranched C1-C6alkyl which is unsubstituted or substituted with one or more of —OH,—SH, —NH₂ or a halogen;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct, as for example, —CN,—CH═NR₅,

R₁ represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group;

R_(3a) represents a hydrogen or a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R_(3b) is absent, or represents a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R_(4a) and R_(4b) each independently represent a hydrogen, lower alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl,carboxyl, carboxamide, carbonyl, or cyano, with the caveat that eitherboth or neither of R_(4a) and R_(4b) are hydrogen;

R_(4c) represents a halogen, an amine, an alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl, carboxyl,carboxamide, carbonyl, or cyano;

R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,—(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH, —(CH₂)n-S-alkyl,—(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl, —(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂,—C(O)C(O)OR₇;

R₆ represents, independently for each occurrence, an aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety;

R₇ represents, independently for each occurrence, hydrogen, or an alkyl,alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle moiety;and

Y₁ and Y₂ each independently represent —OH, or a group capable of beinghydrolyzed to a hydroxyl group, including cyclic derivatives where Y₁and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen;

z is zero or an integer in the range of 1 to 3 (preferably 0 or 1); m iszero or an integer in the range of 1 to 8; and n is an integer in therange of 1 to 8.

Yet another aspect of the present invention provides a compoundrepresented by Formula IV:

wherein

A represents a 3-8 membered heterocycle including the N and the Cαcarbon;

B represents a C3-C8 ring, or C7-C14 fused bicyclic or tricyclic ringsystem;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct, as for example, —CN,—CH═NR₅,

R₁ represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group;

R₂ is absent or represents one or more substitutions to the ring A, eachof which can independently be a halogen, a lower alkyl, a lower alkenyl,a lower alkynyl, a carbonyl (such as a carboxyl, an ester, a formate, ora ketone), a thiocarbonyl (such as a thioester, a thioacetate, or athioformate), an amino, an acylamino, an amido, a cyano, a nitro, anazido, a sulfate, a sulfonate, a sulfonamido, —(CH₂)_(m)—R₆,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₆, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₆;

R_(3b) is absent, or represents a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,—(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH, —(CH₂)n-S-alkyl,—(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl, —(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂,—C(O)C(O)OR₇;

R₆ represents, independently for each occurrence, an aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety;

R₇ represents, independently for each occurrence, hydrogen, or an alkyl,alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle moiety;and

Y₁ and Y₂ each independently represent —OH, or a group capable of beinghydrolyzed to a hydroxyl group, including cyclic derivatives where Y₁and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In certain embodiments, the protease inhibitor is represented in thegeneral formula

where B, R₁, R_(3b) and W are as defined above, and p is an integer from1 to 3. In certain preferred embodiments, p is 1, and R_(3a) is ahydrogen and R_(3b) is absent.

Another aspect of the present invention is a compound represented byFormula VI:

R represents hydrogen, a halogen, or a branched or unbranched C1-C6alkyl which is unsubstituted or substituted with one or more of —OH,—SH, —NH₂ or a halogen;

B represents a C3-C8 ring, or C7-C14 fused bicyclic or tricyclic ringsystem;

W represents a functional group which reacts with an active site residueof the targeted protease to form a covalent adduct, as for example, —CN,—CH═NR₅,

R₁ represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group;

R_(3b) is absent, or represents a substituent which does not conjugatethe electron pair of the nitrogen from which it pends, such as a loweralkyl;

R₅ represents H, an alkyl, an alkenyl, an alkynyl, —C(X₁)(X₂)X₃,—(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl, —(CH₂)n-O-alkenyl,—(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH, —(CH₂)n-S-alkyl,—(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl, —(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂,—C(O)C(O)OR₇;

R₆ represents, independently for each occurrence, an aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety;

R₇ represents, independently for each occurrence, hydrogen, or an alkyl,alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle moiety;and

Y₁ and Y₂ each independently represent —OH, or a group capable of beinghydrolyzed to a hydroxyl group, including cyclic derivatives where Y₁and Y₂ are connected via a ring having from 5 to 8 atoms in the ringstructure (such as pinacol or the like),

R₅₀ represents O or S;

R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇;

R₅₂ represents hydrogen, a lower alkyl, an amine, —OR₇, or apharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with thephosphorous atom to which they are attached complete a heterocyclic ringhaving from 5 to 8 atoms in the ring structure

X₁ represents a halogen;

X₂ and X₃ each represent a hydrogen or a halogen;

m is zero or an integer in the range of 1 to 8; and n is an integer inthe range of 1 to 8.

In certain preferred embodiments of the subject inhibitor structuresabove, W represents:

In certain preferred embodiments of the subject inhibitor structuresabove, R₅ is a hydrogen or —C(X₁)(X₂)X₃, wherein X₁ is a fluorine, andX₂ and X₃, if halogens, are also fluorine.

In certain preferred embodiments of the subject inhibitor structuresabove, R₁ is a peptidyl moiety which is a substrate for a protease whichcleaves between R₁ and its pendent amine moiety. In other preferredembodiments, R₁ is an amino blocking group.

In certain preferred embodiments, A is a 4-8 membered ring, morepreferably a 5, 6 or 7 membered ring. A can be a ring selected from thegroup consisting of azaridines, thiazoles, pyrroles, diazoles (such asimidazoles and pyrazolidines), pyridines, oxazoles, isozazoles,isothiazoles, azepines, diazepines, oxadiazoles, oxatriazoles,dioxazoles, oxathiazoles, pyrimidines, pyridazines, pyranzines,triazines, oxazines, isoxzaines, and oxathiazines, or reduced formsthereof (e.g., dihydro- and tetrahydro-versions thereof), suchpyrrolidines, piperidines, piperazines, morpholines, thiazolidines, andimidazolines.

In certain preferred embodiments, A is a thiazole, pyrrole, or pyridine,or reduced form thereof.

In certain preferred embodiments, R represents hydrogen or a branched orunbranched C1-C6 alkyl;

In certain preferred embodiments, R₂ is absent. In other preferredembodiments, R₂ represents one or two, preferably one, hydroxyl group.

In certain preferred embodiments, R_(3a) and R_(3b) each independentlyrepresent hydrogen. In other preferred embodiments, R_(3a) and R_(3b)each independently represent hydrogen or a C1-C3 alkyl.

In certain preferred embodiments, each of R_(4a) and R_(4b) eachindependently represent (subject to the above proviso) hydrogen or asmall hydrophobic group such as a halogen, a lower alkyl, a loweralkenyl, or a lower alkynyl; and R_(4c) represents a halogen, a loweralkyl, a lower alkenyl, or a lower alkynyl. In certain preferredembodiments, R_(4a) is a hydrogen, and R_(4b) and R_(4c) are both C1-4alkyls, or R_(4a), R_(4b) and R_(4c) are all C1-4 alkyls.

In certain preferred embodiments, R_(4a) and R_(4b) are both hydrogen,and R_(4c) represents a cycloalkyl, a heterocycloalkyl, an aryl orheteroaryl group, such as a 3-8 membered ring, more preferably a 5, 6 or7 membered ring. The ring may be substituted by up to 4heteroatoms—selected from the group consisting of O (oxygen), S(sulphur) or N (nitrogen). In certain preferred embodiments, R_(4c) is acycloalkyl.

In certain embodiments, B is 3-8 membered ring, more preferably a 5, 6or 7 membered ring. B can be a ring selected from the group consistingof azaridines, thiazoles, pyrroles, diazoles (such as imidazoles andpyrazolidines), pyridines, oxazoles, isozazoles, isothiazoles, azepines,diazepines, oxadiazoles, oxatriazoles, dioxazoles, oxathiazoles,pyrimidines, pyridazines, pyranzines, triazines, oxazines, isoxzaines,and oxathiazines, or reduced forms thereof (e.g., dihydro- andtetrahydro-versions thereof), such pyrrolidines, piperidines,piperazines, morpholines, thiazolidines, and imidazolines.

In certain embodiments, B is a bicyclic or tricyclic ring such as anindole, an indolenine, an isobenzazole, a pyrindine, a pyrannopyrrole,an isoindazole, an indoxazine, a benzoxazole, an anthanil, a quinoline,an isoquinoline, a cinnoline, a quinazoline, a napthyridine, apyridopyridine, a benzoxazine, a benzisoxazine, a carbazole, anacridine, or a purine, or reduced forms thereof (e.g., dihydro- andtetrahydro-versions thereof). In certain preferred embodiments, B is atetrahydroisoquinoline or a tetrahydrocarboline (such as a β orγ-carboline).

In certain embodiments, B is unsubstituted, or is substituted with oneor more of —OH, —SH, —NH₂, halogens, or lower alkyl. In certainpreferred embodiments, B is unsubstituted.

In certain embodiments, B is 3-8 membered ring, more preferably a 5, 6or 7 membered ring. B can be a ring selected from the group consistingof azaridines, thiazoles, pyrroles, diazoles (such as imidazoles andpyrazolidines), pyridines, oxazoles, isozazoles, isothiazoles, azepines,diazepines, oxadiazoles, oxatriazoles, dioxazoles, oxathiazoles,pyrimidines, pyridazines, pyranzines, triazines, oxazines, isoxzaines,and oxathiazines, or reduced forms thereof (e.g., dihydro- andtetrahydro-versions thereof), such pyrrolidines, piperidines,piperazines, morpholines, thiazolidines, and imidazolines.

In certain embodiments, B is a bicyclic or tricyclic ring such as anindole, an indolenine, an isobenzazole, a pyrindine, a pyrannopyrrole,an isoindazole, an indoxazine, a benzoxazole, an anthanil, a quinoline,an isoquinoline, a cinnoline, a quinazoline, a napthyridine, apyridopyridine, a benzoxazine, a benzisoxazine, a carbazole, anacridine, or a purine, or reduced forms thereof (e.g., dihydro- andtetrahydro-versions thereof). In certain preferred embodiments, B is atetrahydroisoquinoline or a tetrahydrocarboline (such as a β orγ-carboline).

In certain preferred embodiments, if A represents a pyrrolidine ring,then R_(4a), R_(4b) and R_(4c) are selected such that they do not giverise to a naturally occurring amino acid side chain, e.g., as defined bythe IUPAC-IUB Commission on Biochemical Nomenclature.

In certain preferred embodiments, if R_(4a), R_(4b) and R_(4c) areselected to give rise to a naturally occurring amino acid side chain,e.g., as defined by the IUPAC-IUB Commission on BiochemicalNomenclature, then A is not a pyrrolidine ring.

In certain preferred embodiments, z is zero or 1.

Exemplary structures include compounds include.

In certain preferred embodiments, the subject inhibitors are DPIVinhibitors with a Ki for DPIV inhibition of 10 nm or less, morepreferably of 1.0 nm or less, and even more preferably of 0.1 or even0.01 nM or less. Indeed, inhibitors with Ki values in the picomolar andeven femtomolar range are contemplated.

In general, the inhibitors of the subject method will be smallmolecules, e.g., with molecular weights less than 7500 amu, preferablyless than 5000 amu, and even more preferably less than 2000 amu and even1000 amu. In preferred embodiments, the inhibitors will be orallyactive.

Another aspect of the present invention relates to pharmaceuticalcompositions of dipeptidylpeptidase inhibitors, particularlyinhibitor(s), and their uses in treating and/or preventing disorderswhich can be improved by altering the homeostasis of peptide hormones.In a preferred embodiment, the inhibitors have hypoglycemic andantidiabetic activities, and can be used in the treatment of disordersmarked by abberrant glucose metabolism (including storage). Inparticular embodiments, the compositions of the subject methods areuseful as insulinotropic agents, or to potentiate the insulinotropiceffects of such molecules as GLP-1. In this regard, the present methodcan be useful for the treatment and/or prophylaxis of a variety ofdisorders, including one or more of: hyperlipemia, hyperglycemia,obesity, glucose tolerance insufficiency, insulin resistance anddiabetic complications.

For instance, in certain embodiments the method involves administrationof an inhibitor(s), preferably at a predetermined time(s) during a24-hour period, in an amount effective to improve one or more aberrantindices associated with glucose metabolism disorders (e.g., glucoseintolerance, insulin resistance, hyperglycemia, hyperinsulinemia andType II diabetes). The effective amount of the inhibitor may be about0.01, 0.1, 1, 10, 30, 50, 70, 100, 150, 200, 500, or 1000 mg/kg of thesubject.

(ii). Agonism of GLP-1 Effects

The inhibitors useful in the subject methods possess, in certainembodiments, the ability to lower blood glucose levels, to relieveobesity, to alleviate impaired glucose tolerance, to inhibit hepaticglucose neogenesis, and to lower blood lipid levels and to inhibitaldose reductase. They are thus useful for the prevention and/or therapyof hyperglycemia, obesity, hyperlipidemia, diabetic complications(including retinopathy, nephropathy, neuropathy, cataracts, coronaryartery disease and arteriosclerosis) and furthermore for obesity-relatedhypertension and osteoporosis.

Diabetes mellitus is a disease characterized by hyperglycemia occurringfrom a relative or absolute decrease in insulin secretion, decreasedinsulin sensitivity or insulin resistance. The morbidity and mortalityof this disease result from vascular, renal, and neurologicalcomplications. An oral glucose tolerance test is a clinical test used todiagnose diabetes. In an oral glucose tolerance test, a patient'sphysiological response to a glucose load or challenge is evaluated.After ingesting the glucose, the patient's physiological response to theglucose challenge is evaluated. Generally, this is accomplished bydetermining the patient's blood glucose levels (the concentration ofglucose in the patient's plasma, serum or whole blood) for severalpredetermined points in time.

In one embodiment, the present invention provides a method for agonizingthe action of GLP-1. It has been determined that isoforms ofGLP-1(GLP-1(7-37) and GLP-1(7-36)), which are derived frompreproglucagon in the intestine and the hind brain, have insulinotropicactivity, i.e., they modulate glucose metabolism. DPIV cleaves theisoforms to inactive peptides. Thus, in certain embodiments,inhibitor(s) of the present invention can agonize insulinotropicactivity by interfering with the degradation of bioactive GLP-1peptides.

(iii). Agonism of the Effects of Other Peptide Hormones

In another embodiment, the subject agents can be used to agonize (e.g.,mimic or potentiate) the activity of peptide hormones, e.g., GLP-2, GIPand NPY.

To illustrate further, the present invention provides a method foragonizing the action of GLP-2. It has been determined that GLP-2 acts asa trophic agent, to promote growth of gastrointestinal tissue. Theeffect of GLP-2 is marked particularly by increased growth of the smallbowel, and is therefore herein referred to as an “intestinotrophic”effect. DPIV is known to cleave GLP-2 into a biologically inactivepeptide. Thus, in one embodiment, inhibition of DPIV interferes with thedegradation of GLP-2, and thereby increases the plasma half-life of thathormone.

In still other embodiments, the subject method can be used to increasethe half-life of other proglucagon-derived peptides, such as glicentin,oxyntomodulin, glicentin-related pancreatic polypeptide (GRPP), and/orintervening peptide-2 (IP-2). For example, glicentin has beendemonstrated to cause proliferation of intestinal mucosa and alsoinhibits a peristalsis of the stomach, and has thus been elucidated asuseful as a therapeutic agent for digestive tract diseases, thus leadingto the present invention.

Thus, in one aspect, the present invention relates to therapeutic andrelated uses of inhibitor(s) for promoting the growth and proliferationof gastrointestinal tissue, most particularly small bowel tissue. Forinstance, the subject method can be used as part of a regimen fortreating injury, inflammation or resection of intestinal tissue, e.g.,where enhanced growth and repair of the intestinal mucosal epithelial isdesired.

With respect to small bowel tissue, such growth is measured convenientlyas a increase in small bowel mass and length, relative to an untreatedcontrol. The effect of subject inhibitors on small bowel also manifestsas an increase in the height of the crypt plus villus axis. Suchactivity is referred to herein as an “intestinotrophic” activity. Theefficacy of the subject method may also be detectable as an increase incrypt cell proliferation and/or a decrease in small bowel epitheliumapoptosis. These cellular effects may be noted most significantly inrelation to the jejunum, including the distal jejunum and particularlythe proximal jejunum, and also in the distal ileum. A compound isconsidered to have “intestinotrophic effect” if a test animal exhibitssignificantly increased small bowel weight, increased height of thecrypt plus villus axis, or increased crypt cell proliferation ordecreased small bowel epithelium apoptosis when treated with thecompound (or genetically engineered to express it themselves). A modelsuitable for determining such gastrointestinal growth is described byU.S. Pat. No. 5,834,428.

In general, patients who would benefit from either increased smallintestinal mass and consequent increased small bowel mucosal functionare candidates for treatment by the subject method. Particularconditions that may be treated include the various forms of sprueincluding celiac sprue which results from a toxic reaction to O-gliadinfrom wheat, and is marked by a tremendous loss of villae of the bowel;tropical sprue which results from infection and is marked by partialflattening of the villae; hypogammaglobulinemic sprue which is observedcommonly in patients with common variable immunodeficiency orhypogammaglobulinemia and is marked by significant decrease in villusheight. The therapeutic efficacy of the treatment may be monitored byenteric biopsy to examine the villus morphology, by biochemicalassessment of nutrient absorption, by patient weight gain, or byamelioration of the symptoms associated with these conditions. Otherconditions that may be treated by the subject method, or for which thesubject method may be useful prophylactically, include radiationenteritis, infectious or post-infectious enteritis, regional enteritis(Crohn's disease), small intestinal damage due to toxic or otherchemotherapeutic agents, and patients with short bowel syndrome.

More generally, the present invention provides a therapeutic method fortreating digestive tract diseases. The term “digestive tract” as usedherein means a tube through which food passes, including stomach andintestine. The term “digestive tract diseases” as used herein meansdiseases accompanied by a qualitative or quantitative abnormality in thedigestive tract mucosa, which include, e. g., ulceric or inflammatorydisease; congenital or acquired digestion and absorption disorderincluding malabsorption syndrome; disease caused by loss of a mucosalbarrier function of the gut; and protein-losing gastroenteropathy. Theulceric disease includes, e.g., gastric ulcer, duodenal ulcer, smallintestinal ulcer, colonic ulcer and rectal ulcer. The inflammatorydisease include, e.g., esophagitis, gastritis, duodenitis, enteritis,colitis, Crohn's disease, proctitis, gastrointestinal Behcet, radiationenteritis, radiation colitis, radiation proctitis, enteritis andmedicamentosa. The malabsorption syndrome includes the essentialmalabsorption syndrome such as disaccharide-decomposing enzymedeficiency, glucose-galactose malabsorption, fractose malabsorption;secondary malabsorption syndrome, e.g., the disorder caused by a mucosalatrophy in the digestive tract through the intravenous or parenteralnutrition or elemental diet, the disease caused by the resection andshunt of the small intestine such as short gut syndrome, cul-de-sacsyndrome; and indigestible malabsorption syndrome such as the diseasecaused by resection of the stomach, e.g., dumping syndrome.

The term “therapeutic agent for digestive tract diseases” as used hereinmeans the agents for the prevention and treatment of the digestive tractdiseases, which include, e.g., the therapeutic agent for digestive tractulcer, the therapeutic agent for inflammatory digestive tract disease,the therapeutic agent for mucosal atrophy in the digestive tract and thetherapeutic agent for digestive tract wound, the amelioration agent forthe function of the digestive tract including the agent for recovery ofthe mucosal barrier function and the amelioration agent for digestiveand absorptive function. The ulcers include digestive ulcers anderosions, acute ulcers, namely, acute mucosal lesions.

The subject method, because of promoting proliferation of intestinalmucosa, can be used in the treatment and prevention of pathologicconditions of insufficiency in digestion and absorption, that is,treatment and prevention of mucosal atrophy, or treatment of hypoplasiaof the digestive tract tissues and decrease in these tissues by surgicalremoval as well as improvement of digestion and absorption. Further, thesubject method can be used in the treatment of pathologic mucosalconditions due to inflammatory diseases such as enteritis, Crohn'sdisease and ulceric colitis and also in the treatment of reduction infunction of the digestive tract after operation, for example, in dampingsyndrome as well as in the treatment of duodenal ulcer in conjunctionwith the inhibition of peristalsis of the stomach and rapid migration offood from the stomach to the jejunum.

Furthermore, glicentin can effectively be used in promoting cure ofsurgical invasion as well as in improving functions of the digestivetract. Thus, the present invention also provides a therapeutic agent foratrophy of the digestive tract mucosa, a therapeutic agent for wounds inthe digestive tract and a drug for improving functions of the digestivetract which comprise glicentin as active ingredients.

Likewise, the inhibitor(s) of the subject invention can be used to alterthe plasma half-life of secretin, VIP, PHI, PACAP, GIP and/orhelodermin. Additionally, the subject method can be used to alter thepharmacokinetics of Peptide YY and neuropeptide Y, both members of thepancreatic polypeptide family, as DPIV has been implicated in theprocessing of those peptides in a manner which alters receptorselectivity.

Neuropeptide Y (NPY) is believed to act in the regulation vascularsmooth muscle tone, as well as regulation of blood pressure. NPY alsodecreases cardiac contractility. NPY is also the most powerful appetitestimulant known (Wilding et al., (1992) J Endocrinology 132:299-302).The centrally evoked food intake (appetite stimulation) effect ispredominantly mediated by NPY Y1 receptors and causes increase in bodyfat stores and obesity (Stanley et al., (1989) Physiology and Behavior46:173-177).

According to the present invention, a method for treatment of anorexiacomprises administering to a host subject an effective amount of aninhibitor(s) to stimulate the appetite and increase body fat storeswhich thereby substantially relieves the symptoms of anorexia.

A method for treatment of hypotension comprises administering to a hostsubject an effective amount of an inhibitor(s) of the present inventionto mediate vasoconstriction and increase blood pressure which therebysubstantially relieves the symptoms of hypotension.

DPIV has also been implicated in the metabolism and inactivation ofgrowth hormone-releasing factor (GHRF). GHRF is a member of the familyof homologous peptides that includes glucagon, secretin, vasoactiveintestinal peptide (VIP), peptide histidine isoleucine (PHI), pituitaryadenylate cyclase activating peptide (PACAP), gastric inhibitory peptide(GIP) and helodermin. Kubiak et al. (1994) Peptide Res 7:153. GHRF issecreted by the hypothalamus, and stimulates the release of growthhormone (GH) from the anterior pituitary. Thus, the subject method canbe used to improve clinical therapy for certain growth hormone deficientchildren, and in clinical therapy of adults to improve nutrition and toalter body composition (muscle vs. fat). The subject method can also beused in veterinary practice, for example, to develop higher yield milkproduction and higher yield, leaner livestock.

(iv). Assays of Insulinotropic Activity

In selecting a compound suitable for use in the subject method, it isnoted that the insulinotropic property of a compound may be determinedby providing that compound to animal cells, or injecting that compoundinto animals and monitoring the release of immunoreactive insulin (IRI)into the media or circulatory system of the animal, respectively. Thepresence of IRI can be detected through the use of a radioimmunoassaywhich can specifically detect insulin.

The db/db mouse is a genetically obese and diabetic strain of mouse. Thedb/db mouse develops hyperglycemia and hyperinsulinemia concomitant withits development of obesity and thus serves as a model of obese type 2diabetes (NIDDM). The db/db mice can purchased from, for example, TheJackson Laboratories (Bar Harbor, Me.). In an exemplary embodiment, fortreatment of the mice with a regimen including an inhibitor(s) orcontrol, sub-orbital sinus blood samples are taken before and at sometime (e.g., 60 minutes) after dosing of each animal. Blood glucosemeasurements can be made by any of several conventional techniques, suchas using a glucose meter. The blood glucose levels of the control andinhibitor(s) dosed animals are compared

The metabolic fate of exogenous GLP-1 can also be followed in eithernondiabetic and type II diabetic subjects, and the effect of a candidateinhibitor(s) determined. For instance, a combination of high-pressureliquid chromatography (HPLC), specific radioimmunoassays (RIAs), and aenzyme-linked immunosorbent assay (ELISA), can be used, whereby intactbiologically active GLP-1 and its metabolites can be detected. See, forexample, Deacon et al. (1995) Diabetes 44:1126-1131. To illustrate,after GLP-1 administration, the intact peptide can be measured using anNH2-terminally directed RIA or ELISA, while the difference inconcentration between these assays and a COOH-terminal-specific RIAallowed determination of NH2-terminally truncated metabolites. Withoutinhibitor, subcutaneous GLP-1 is rapidly degraded in a time-dependentmanner, forming a metabolite which co-elutes on HPLC with GLP-I(9-36)amide and has the same immunoreactive profile. For instance, thirtyminutes after subcutaneous GLP-1 administration to diabetic patients(n=8), the metabolite accounted for 88.5+1.9% of the increase in plasmaimmunoreactivity determined by the COOH-terminal RIA, which was higherthan the levels measured in healthy subjects (78.4+3.2%; n=8; P<0.05).See Deacon et al., supra. Intravenously infused GLP-I was alsoextensively degraded.

(v). Conjoint Administration

Another aspect of the invention provides a conjoint therapy wherein oneor more other therapeutic agents are administered with the proteaseinhibitor. Such conjoint treatment may be achieved by way of thesimultaneous, sequential or separate dosing of the individual componentsof the treatment.

In one embodiment, an inhibitor(s) is conjointly administered withinsulin or other insulinotropic agents, such as GLP-1, peptide hormones,such as GLP-2, GIP, or NPY, or a gene therapy vector which causes theectopic expression of said agents and peptide hormones. In certainembodiments, said agents or peptide hormones may be variants of anaturally occurring or synthetic peptide hormone, wherein one or moreamino acids have been added, deleted or substituted.

In another illustrative embodiment, the subject inhibitors can beconjointly administered with a an M1 receptor antagonist. Cholinergicagents are potent modulators of insulin release that act via muscarinicreceptors. Moreover, the use of such agents can have the added benefitof decreasing cholesterol levels, while increasing HDL levels. Suitablemuscarinic receptor antagonists include substances that directly orindirectly block activation of muscarinic cholinergic receptors.Preferably, such substances are selective (or are used in amounts thatpromote such selectivity) for the M1 receptor. Nonlimiting examplesinclude quaternary amines (such as methantheline, ipratropium, andpropantheline), tertiary amines (e.g. dicyclomine, scopolamine) andtricyclic amines (e.g. telenzepine). Pirenzepine and methyl scopolamineare preferred. Other suitable muscarinic receptor antagonists includebenztropine (commercially available as COGENTIN from Merck),hexahydro-sila-difenidol hydrochloride (HHSID hydrochloride disclosed inLambrecht et al. (1989) Trends in Pharmacol. Sci. 10(Suppl):60;(+/−)-3-quinuclidinyl xanthene-9-carboxylate hemioxalate(QNX-hemioxalate; Birdsall et al., Trends in Pharmacol. Sci. 4:459,1983; telenzepine dihydrochloride (Coruzzi et al. (1989) Arch. Int.Pharmacodyn. Ther. 302:232; and Kawashima et al. (1990) Gen. Pharmacol.21:17) and atropine. The dosages of such muscarinic receptor antagonistswill be generally subject to optimization as outlined above. In the caseof lipid metabolism disorders, dosage optimization may be necessaryindependently of whether administration is timed by reference to thelipid metabolism responsiveness window or not.

In terms of regulating insulin and lipid metabolism and reducing theforegoing disorders, the subject inhibitor(s) may also actsynergistically with prolactin inhibitors such as d2 dopamine agonists(e.g. bromocriptine). Accordingly, the subject method can include theconjoint administration of such prolactin inhibitors asprolactin-inhibiting ergo alkaloids and prolactin-inhibiting dopamineagonists. Examples of suitable compounds include2-bromo-alpha-ergocriptine, 6-methyl-8beta-carbobenzyloxyaminoethyl-10-alpha-ergoline, 8-acylaminoergolines,6-methyl-8-alpha-(N-acyl)amino-9-ergoline,6-methyl-8-alpha-(N-phenylacetyl)amino-9-ergoline, ergocornine,9,10-dihydroergocornine, D-2-halo-6-alkyl-8-substituted ergolines,D-2-bromo-6-methyl-8-cyanomethylergoline, carbidopa, benserazide andother dopadecarboxylase inhibitors, L-dopa, dopamine and non toxic saltsthereof.

The inhibitor(s) used according to the invention can also be usedconjointly with agents acting on the ATP-dependent potassium channel ofthe β-cells, such as glibenclamide, glipizide, gliclazide and AG-EE 623ZW. The inhibitor(s) may also advantageously be applied in combinationwith other oral agents such as metformin and related compounds orglucosidase inhibitors as, for example, acarbose.

(vi). Pharmaceutical Compositions

Inhibitors prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, conditionand body weight of the patient, as is well known in the art. Forexample, where the compounds are to be administered orally, they may beformulated as tablets, capsules, granules, powders or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular or subcutaneous), drop infusion preparationsor suppositories. For application by the ophthalmic mucous membraneroute, they may be formulated as eyedrops or eye ointments. Theseformulations can be prepared by conventional means, and, if desired, theactive ingredient may be mixed with any conventional additive, such asan excipient, a binder, a disintegrating agent, a lubricant, acorrigent, a solubilizing agent, a suspension aid, an emulsifying agentor a coating agent. Although the dosage will vary depending on thesymptoms, age and body weight of the patient, the nature and severity ofthe disorder to be treated or prevented, the route of administration andthe form of the drug, in general, a daily dosage of from 0.01 to 2000 mgof the compound is recommended for an adult human patient, and this maybe administered in a single dose or in divided doses.

The precise time of administration and/or amount of the inhibitor thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of a particular compound, physiological condition of thepatient (including age, sex, disease type and stage, general physicalcondition, responsiveness to a given dosage and type of medication),route of administration, etc. However, the above guidelines can be usedas the basis for fine-tuning the treatment, e.g., determining theoptimum time and/or amount of administration, which will require no morethan routine experimentation consisting of monitoring the subject andadjusting the dosage and/or timing.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting the subject chemical fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The term “pharmaceutically acceptable salts” refers to the relativelynon-toxic, inorganic and organic acid addition salts of theinhibitor(s). These salts can be prepared in situ during the finalisolation and purification of the inhibitor(s), or by separatelyreacting a purified inhibitor(s) in its free base form with a suitableorganic or inorganic acid, and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate,stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19)

In other cases, the inhibitors useful in the methods of the presentinvention may contain one or more acidic functional groups and, thus,are capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable bases. The term “pharmaceutically acceptablesalts” in these instances refers to the relatively non-toxic, inorganicand organic base addition salts of an inhibitor(s). These salts canlikewise be prepared in situ during the final isolation and purificationof the inhibitor(s), or by separately reacting the purified inhibitor(s)in its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like(see, for example, Berge et al., supra).

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations useful in the methods of the present invention includethose suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal, aerosol and/or parenteral administration.The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

Methods of preparing these formulations or compositions include the stepof bringing into association an inhibitor(s) with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a ligand with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or nonaqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes and the like, each containinga predetermined amount of an inhibitor(s) as an active ingredient. Acompound may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules and the like), the active ingredient is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, acetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered peptide orpeptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active ingredient, the liquid dosage formsmay contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethyleneglycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of aninhibitor(s) include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active componentmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams and gels may contain, in addition toinhibitor(s), excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an inhibitor(s),excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The inhibitor(s) can be alternatively administered by aerosol. This isaccomplished by preparing an aqueous aerosol, liposomal preparation orsolid particles containing the compound. A nonaqueous (e.g.,fluorocarbon propellant) suspension could be used. Sonic nebulizers arepreferred because they minimize exposing the agent to shear, which canresult in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueoussolution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular compound, buttypically include nonionic surfactants (Tweens, Pluronics, orpolyethylene glycol), innocuous proteins like serum albumin, sorbitanesters, oleic acid, lecithin, amino acids such as glycine, buffers,salts, sugars or sugar alcohols. Aerosols generally are prepared fromisotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of an inhibitor(s) to the body. Such dosage forms can be madeby dissolving or dispersing the agent in the proper medium. Absorptionenhancers can also be used to increase the flux of the inhibitor(s)across the skin. The rate of such flux can be controlled by eitherproviding a rate controlling membrane or dispersing the peptidomimeticin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like,are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteraladministration comprise one or more inhibitors(s) in combination withone or more pharmaceutically acceptable sterile isotonic aqueous ornonaqueous solutions, dispersions, suspensions or emulsions, or sterilepowders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofinhibitor(s) in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

When the inhibitors(s) of the present invention are administered aspharmaceuticals, to humans and animals, they can be given per se or as apharmaceutical composition containing, for example, 0.1 to 99.5% (morepreferably, 0.5 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are of course given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. Oral administration is preferred.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug or other material otherthan directly into the central nervous system, such that it enters thepatient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These inhibitors(s) may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,nasally, as by, for example, a spray, rectally, intravaginally,parenterally, intracisternally and topically, as by powders, ointmentsor drops, including buccally and sublingually.

Regardless of the route of administration selected, the inhibitor(s),which may be used in a suitable hydrated form, and/or the pharmaceuticalcompositions of the present invention, are formulated intopharmaceutically acceptable dosage forms by conventional methods knownto those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

IV. Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Abbreviations

EDC: N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride;

HOBT: 1-Hydroxybenzotriazole;

Chg: Cyclohexylglycine.

Example 1: Synthesis of Cyclohexylglycine boroAla

Referring to FIG. 1, a solution of 515 mg (2.00 mmol) ofBoc-L-2-(cyclohexyl)glycine 1 (Chem-Impex International), 587 mg (2.26mmol) of HCl.boroAla pinane 2, 332 mg (2.46 mmol) of HOBT, and 671 μL(4.84 mmol) of triethylamine in 6 mL of anhydrous DMF was treated with498 mg (2.60 mmol) of EDC, and the resulting solution stirred at roomtemperature under argon for 18 h. The reaction mixture was diluted witha 200 mL of 10% aqueous citric acid and the resulting mixture extractedwith 2×100 mL of ethyl acetate. The combined extracts were washed withbrine, dried (MgSO₄), filtered, and concentrated to give a clear oil.The crude oil was chromatographed over silica gel with ethylacetate/hexane to give the product ester as a clear oil. The oil wasthen dissolved in hydrogen chloride in diethyl ether (1.0 M solution, 25mL) and stirred for 48 hours at room temperature. The mixture wasevaporated to dryness in vacuo and redissolved in 25 mL phenylboronicacid solution (244 mg, 2 mmol) at pH 2 (0.01 N HCl) and ether (25 mL).After stirring for 30 min, the ether layer was removed and replaced withfresh ether (25 mL). This step was repeated for four times. The aqueousphase was then lyophilized and purified by HPLC to afford 170 mg (37%)of the target compound 3.

Example 2: Glucose Tolerance Test

Experiments show that cyclohexyl-gly boro ala is orally active andclearly lowers blood sugar based upon results from an oral glucosechallenge in zucker obese rats. See FIG. 2. In these “acute” experimentszucker obese and zucker lean rats were orally administered either 0.035mg/kg (low dose) or 0.35 mg/kg (high dose) of cyclohexyl-gly boro alaand then subjected to an oral glucose tolerance test within an hour.Each set of experiments was also performed using saline as a control.

Example 3: Inhibitor Inactivation at pH 8

Experiments show that Cyclohexylglycine-bAla does not show significantpH-time dependant inhibition as compared with His-bAla, Ala-bAla, andPhg-bAla. In this experiment stock solutions of inhibitors (His-bAla,Ala-bAla, Phg-bAla and Cyclohexylglycine-bAla) were prepared at pH 1-2.These stock solutions were pre-incubated at pH 8 as follows: first, 1:10dilutions into a buffer (0.1 M HEPES pH 8, 0.14 M NaCl) were performed;secondly, the pH was measured after dilution and varied for differentinhibitors between 7.5 and 8; and thirdly, incubations at this pH wereperformed for 0, 60, 120, 180 minutes. Following incubation, 1:10 serialdilutions of inhibitors in buffer and 1:10 dilution of inhibitors intoEnzyme (DPPIV) in buffer were made. The inhibitors were pre-incubatedwith enzyme for 10 minutes to account for slow binding and substrate(H-Ala-Pro-paranitoranalide) was added at a concentration of approx.=KM(17 μM). Absorbances at 410 nm were recorded for all inhibitors after 30minutes. See FIGS. 3-6.

Example 4: DPPIV Assays on Serum Samples from Rats

Experiments show that DPPIV enzyme activity was significantly decreasedin rats treated with Cyclohexylglycine-boroAla. See FIG. 7. Four ratswere used in this experiment: two females (#3 and #9) and two males (#10and #11). Blood and plasma samples were collected from rats 1 hour afterbeing treated with Cyclohexylglycine-boroAla. The collected serumsamples were evaluated for DPPIV activity of Cyclohexylglycine-boroAlaas follows:

-   -   2 mg of Ala-Pro-paranitroanalide (substrate) was dissolved in 20        ml 0.1 M HEPES pH 8, 0.14 M NaCl (buffer).    -   Serum samples were diluted into substrate solution in the wells        of a microtiter plate. For each sample, 10 uL of serum was        diluted into 150 μL of substrate.    -   A reading of the A410 in each well was recorded immediately        after the dilution of serum into substrate, and again after        approximately 1 hour. The time of data acquisition for each        reading is recorded in the data file by the microplate reader        software.

The rate of absorbance change was obtained by subtracting the firstreading from the second and dividing by the reaction time to giveDeltaA410/hr. The DPPIV activity was plotted in units of DeltaA410 hr⁻¹μL⁻¹.

Example 5: Prevention of Cyclization by Using Bulky Substituents

In this example, Xaa-boro-Ala analogs containing bulky R substituentwill be constructed to prevent cyclization and increase biologicalactivity. See FIG. 8. The inventors have previously shown that syntheticdiastereomeric monomeric compounds, e.g., L-Ala-D,L-boroPro andL-Pro-D,L-boroPro, were potent inhibitors of the catalytic activity ofsoluble DPIV. They also encountered a problem because these monomericinhibitors lost some of their inhibitory activity rapidly in aqueoussolution at pH value around neutral due to a cyclization reaction. Theopen, active, inhibitory chain species is favored at low pH while thecyclized structure is favored at high pH. Also, the reaction is fullyreversible: the open chain becomes predominate at low pH. The open chainto cyclic species reaction involves a trans to cis isomerization of theproline and the formation of a new N—B bond. The half life for thereformation of the open chain species from the cyclic structure issurprisingly slow. It has been demonstrated that the ratio of[cyclic]:[open] forms, at neutral pH, is 156:1 for Pro-boroPro and1130:1 for Val-boroPro (W. G. Gutheil and W. W. Bachovchin, Separationof L-Pro-DL-boroPro into Its Component Diastereomers and KineticAnalysis of Their Inhibition of Dipeptidyl Peptidase IV. A New Methodfor the Analysis of Slow, Tight-Binding Inhibition, Biochemistry 32,8723-8731 (1993)). This means that less than 1% Pro-boroPro and lessthan 0.1% of Val-boroPro exists as the open chain, inhibitory species,at equilibrium at pH 7.0.

One feature of the present invention relates to the equilibrium constantfor cyclization. It has been found that the ratio of [cyclic]:[open]forms for Cyclohexylglycine-boro-Ala, at neutral pH, is approximately2:1, which is significantly lower than the corresponding ratio forXaa-boro-Pro, as previously disclosed. In addition, the cis-transisomerization rate, and therefore the rates of cyclization anduncyclization, are also much faster for compounds of the presentinvention. This feature is attributed by the inventors to a bulkysubstituent effect, e.g. where R in FIG. 8 represents a cyclohexyl.

The inventors predict that biological bioavailability (biologicalfunction) for the compounds taught in this invention could besignificantly increased (approximately 100-1000 times) by preventingpeptide conformational changes, e.g., intramolecular cyclization, byconstructing compounds bearing a variety of bulky R groups (see FIG. 8).Such compounds include but are not limited to compounds containingunnaturally occurring amino acids at P2.

IV Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All of the above-cited references and publications are herebyincorporated by reference.

1. A protease inhibitor represented by Formula I:

A represents a 3-8 membered heterocycle including the N and the Cαcarbon; W represents a functional group which reacts with an active siteresidue of the targeted protease to form a covalent adduct; R₁represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group; R₂ is absent or representsone or more substitutions to the ring A, each of which can independentlybe a halogen, a lower alkyl, a lower alkenyl, a lower alkynyl, acarbonyl, a thiocarbonyl, an amino, an acylamino, an amido, a cyano, anitro, an azido, a sulfate, a sulfonate, a sulfonamido, —(CH₂)_(m)—R₆,—(CH₂)_(m)—OH, —(CH₂)_(m)—O-lower alkyl, —(CH₂)_(m)—O-lower alkenyl,—(CH₂)_(n)—O—(CH₂)_(m)—R₆, —(CH₂)_(m)—SH, —(CH₂)_(m)—S-lower alkyl,—(CH₂)_(m)—S-lower alkenyl, —(CH₂)_(n)—S—(CH₂)_(m)—R₆; R_(3a) representsa hydrogen or a substituent which does not conjugate the electron pairof the nitrogen from which it pends; R_(3b) is absent, or represents asubstituent which does not conjugate the electron pair of the nitrogenfrom which it pends, such as a lower alkyl; R_(4a) and R_(4b) eachindependently represent a hydrogen, lower alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl, carboxyl,carboxamide, carbonyl, or cyano, with the caveat that either both orneither of R4a and R4b are hydrogen; R_(4c) represents a halogen, anamine, an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, alkoxyl, carboxyl, carboxamide, carbonyl, or cyano; R₆represents, independently for each occurrence, an aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety; z is zero or an integerin the range of 1 to 3; m is zero or an integer in the range of 1 to 8;and n is an integer in the range of 1 to
 8. 2. A protease inhibitorrepresented by Formula III:

wherein R represents hydrogen, a halogen, or a branched or unbranchedC1-C6 alkyl; W represents a functional group which reacts with an activesite residue of the targeted protease to form a covalent adduct; R₁represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group; R_(3a) represents a hydrogenor a substituent which not conjugate the electron pair of the nitrogenfrom which it pends, such as a lower alkyl; R_(3b) is absent, orrepresents a substituent which does not conjugate the electron pair ofthe nitrogen from which it pends, such as a lower alkyl; R_(4a) andR_(4b) each independently represent a hydrogen, lower alkyl,heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxyl,carboxyl, carboxamide, carbonyl, or cyano, with the caveat that eitherboth or neither of R_(4a) and R_(4b) are hydrogen; R_(4c) represents ahalogen, an amine, an alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, alkoxyl, carboxyl, carboxamide, carbonyl, or cyano;and z is zero or an integer in the range of 1 to
 3. 3. A proteaseinhibitor represented by Formula IV: wherein A represents a 3-8 memberedheterocycle including the N and the Ca carbon; B represents a C3-C8ring, or C7-C14 fused bicyclic or tricyclic ring system; W represents afunctional group which reacts with an active site residue of thetargeted protease to form a covalent adduct; R1 represents a hydrogen, aC-terminally linked amino acid or peptide or analog thereof, or aminoprotecting group, R2 is absent or represents one or more substitutionsto the ring A, each of which can independently be a halogen, a loweralkyl, a lower alkenyl, a lower alkynyl, a carbonyl, a thiocarbonyl, anamino, an acylamino, an amido, a cyano, a nitro, an azido, a sulfate, asulfonate, a sulfonamido, —(CH2) m-R6, —(CH2) m-OH, —(CH2) m-0-loweralkyl, —(CH2) m-0-lower alkenyl, —(CH2) n-O—(CH2) m-R6, —(CH2) m-SH,—(CH2) m-S-lower alkyl, —(CH2) m-S-lower alkenyl, —(CH2) ri-S—(CH2)m-R6; R3b is absent, or represents a substituent which does notconjugate the electron pair of the nitrogen from which it pends, such asa lower alkyl; R6 represents, independently for each occurrence, anaryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle moiety; m iszero or an integer in the range of 1 to 8; and n is an integer in therange of 1 to
 8. 4. A protease inhibitor represented by Formula VI:

wherein B represents a C3-C8 ring, or C7-C14 fused bicyclic or tricyclicring system; W represents a functional group which reacts with an activesite residue of the targeted protease to form a covalent adduct; Rrepresents hydrogen, a halogen, or a branched or unbranched C1-C6 alkyl;R₁ represents a hydrogen, a C-terminally linked amino acid or peptide oranalog thereof, or amino protecting group; and R_(3b) is absent, orrepresents a substituent which does not conjugate the electron pair ofthe nitrogen from which it pends, such as a lower alkyl.
 5. Theinhibitor of claim 1, wherein W represents —CN, —CH═NR₅,

wherein, Y₁ and Y₂ each independently represent —OH, or a group capableof being hydrolyzed to a hydroxyl group, including cyclic derivativeswhere Y₁ and Y₂ are connected via a ring having from 5 to 8 atoms in thering structure; R₅ represents H, an alkyl, an alkenyl, an alkynyl,—C(X₁)(X₂)X₃, —(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl,—(CH₂)n-O-alkenyl, —(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH,—(CH₂)n-S-alkyl, —(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl,—(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂, —C(O)C(O)OR₇; R₆ represents,independently for each occurrence, an aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle moiety; R₇ represents, independently foreach occurrence, hydrogen, or an alkyl, alkenyl, aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety; R₅₀ represents O or S;R₅₁ represents N₃, SH₂, NH₂, NO₂ or —OR₇; R₅₂ represents hydrogen, alower alkyl, an amine, —OR₇, or a pharmaceutically acceptable salt, orR₅₁ and R₅₂ taken together with the phosphorous atom to which they areattached complete a heterocyclic ring having from 5 to 8 atoms in thering structure X₁ represents a halogen; X₂ and X₃ each represent ahydrogen or a halogen; m is zero or an integer in the range of 1 to 8;and n is an integer in the range of 1 to
 8. 6. The inhibitor of claim 1,wherein R_(4a), R_(4b) and R_(4c) each independently represent a smallhydrophobic group.
 7. The inhibitor or claim 6, wherein R_(4a), R_(4b)and R_(4c), are independently selected from the group consisting ofhalogens, lower alkyls, lower alkenyls, and lower alkynyls.
 8. Theinhibitor of claim 1, wherein R_(4a) and R_(4b) each represent hydrogen,and R_(4c) represents a small hydrophobic group.
 9. The inhibitor ofclaim 1, wherein R_(4a) and R_(4b) each represent hydrogen, andrepresents a cycloalkyl, heterocycloalkyl, aryl, or heteroaryl.
 10. Theinhibitor of claim 9, wherein R_(4c) represents a C3-C8 cycloalkyl. 11.The inhibitor of claim 1, wherein R₂ is absent, or represents —OH. 12.The inhibitor of claim 1, wherein R_(3a) a hydrogen and R_(3b) isabsent.
 13. The inhibitor of any of claim 1, wherein W represents:

wherein, Y₁ and Y₂ each independently represent —OH, or a group capableof being hydrolyzed to a hydroxyl group, including cyclic derivativeswhere Y₁ and Y₂ are connected via a ring having from 5 to 8 atoms in thering structure; R₅ represents H, an alkyl, an alkenyl, an alkynyl,—C(X₁)(X₂)X₃, —(CH₂)m-R₆, —(CH₂)n-OH, —(CH₂)n-O-alkyl,—(CH₂)n-O-alkenyl, —(CH₂)n-O-alkynyl, —(CH₂)n-O—(CH₂)m-R₆, —(CH₂)n-SH,—(CH₂)n-S-alkyl, —(CH₂)n-S-alkenyl, —(CH₂)n-S-alkynyl,—(CH₂)n-S—(CH₂)m-R₆, —C(O)C(O)NH₂, —C(O)C(O)OR₇; R₆ represents,independently for each occurrence, an aryl, aralkyl, cycloalkyl,cycloalkenyl, or heterocycle moiety; R₇ represents, independently foreach occurrence, hydrogen, or an alkyl, alkenyl, aryl, aralkyl,cycloalkyl, cycloalkenyl, or heterocycle moiety; X₁ represents ahalogen; X₂ and X₃ each represent a hydrogen or a halogen; m is zero oran integer in the range of 1 to 8; and n is an integer in the range of 1to
 8. 14. The inhibitor of claim 13, wherein R₅ is a hydrogen or—C(X₁)(X₂)X₃, wherein X₁ is a fluorine, and X₂ and X₃, if halogens, arealso fluorine.
 15. The inhibitor of claim 1, wherein R₁ is an amino acidresidue or a peptidyl moiety which is a substrate for a protease. 16-17.(canceled)
 18. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a protease inhibitor of claim 1,or a pharmaceutically acceptable salt or prodrug thereof. 19-22.(canceled)
 23. A method for inhibiting the proteolytic activity of apost-proline cleaving enzyme, comprising contacting the enzyme with aprotease inhibitor of claim
 1. 24. A packaged pharmaceutical comprising:a preparation of the protease inhibitor of claim 1; a pharmaceuticallyacceptable carrier; and instructions, written and/or pictorial,describing the use of the preparation for inhibiting a post-prolinecleaving enzyme in vivo.
 25. A packaged pharmaceutical comprising: apreparation of the protease inhibitor of claim 1; a pharmaceuticallyacceptable carrier; and instructions, written and/or pictorial,describing the use of the preparation for regulating glucose metabolism.26. The packaged pharmaceutical of claim 25, wherein the proteaseinhibitor is co-formulated with, or co-packaged with, insulin and/or aninsulinotropic agent.
 27. The packaged pharmaceutical of claim 25,wherein the protease inhibitor is co-formulated with, or co-packagedwith, an MI receptor antagonist, a prolactin inhibitor, agents acting onthe ATP-dependent potassium channel of p-cells, metformin, and/orglucosidase inhibitors.